Advances in rapid isolation techniques and mass spectrometric identification of protein complexes have produced a plethora of protein interaction data. (Gavin et al., Nature 2002, 415, 141-7; Ho et al., Nature 2002, 415, 180-3; Rout et al., J Cell Biol 2000, 148, 635-51; Archambault et al., Cell 2004, 14, 699-711; Sanders et al., Cell Biol 2002, 22, 4723-38.) Researchers are now faced with the challenge of determining which of these interactions are specific or non-specific to the complex under study. Protein complexes are now routinely immunoisolated from cell lysates via an affinity-tagged member of the complex(es). These technologies have become widely utilized in S. cerevisiae due to the relative ease of incorporating a genomic tag by homologous recombination, and also to the commercially available TAP-tag collection of dual affinity-tagged proteins. The immunoisolation technique is an exceptionally powerful method for rapidly and efficiently extracting a protein complex from cell lysate under conditions that preserve in vivo protein interactions.
Nevertheless, a common problem is the co-enrichment of proteins that associate non-specifically with the affinity-tagged complex (e.g., heat-shock or metabolic proteins). In order to attenuate non-specific interactions, researchers typically increase the stringency of the isolation conditions until the non-specific proteins are no longer present. However, it is difficult to determine whether the increased stringency also results in the loss of specific protein-protein interactions.